Project summary/abstract. An adult makes 2.4 million red cells per second and production increases 5-10-fold as the physiological response to anemia. Over 95% of red cell protein content is hemoglobin; each cell contains 270 million hemoglobin molecules; each molecule contains two ? and two ?-globin chains plus four heme moieties; yet free-heme is toxic and must be tightly regulated. As expected from these rapid kinetics, CFU-E/early proerythroblasts are especially vulnerable to heme toxicity since this is when heme synthesis intensifies but globin expression is low. Our previous studies demonstrate that the heme exporter, FLVCR, is critical at this stage and functions as a safety valve, exporting excessive heme. The goals of this competitive renewal application are to study how heme regulates normal red cell differentiation and to determine why excess heme results in cell death. Since heme is synthesized from succinyl CoA (a TCA cycle intermediate) and glycine (an amino acid) and functions as a sensor of metabolic need and protein availability, these data should also provide insight into other quick on-off processes regulated by heme, such as circadian rhythm and N-end rule pathway protein ubiquitination. The observation that anemia occurs when the synthesis of heme in CFU-E/proerythroblasts exceeds its use (in hemoglobin) or export (via FLVCR) also prompts our studies of Diamond Blackfan anemia (DBA) and the myelodysplasia resulting from the isolated deletion of chromosome 5q (del(5q) MDS). These clinical disorders are characterized by haploinsufficiency of ribosomal proteins and poor ribosomal assembly. During the last funding cycle, we showed that although heme synthesis initiates normally, globin translation is slowed. Heme exceeds the capacity of FLVCR and induces excess reactive oxygen species (ROS) and cell death. We propose to investigate the fate of individual early erythroid cells from Flvcr-deleted mice and DBA and MDS patients, using single cell RNA sequencing (in collaboration with Qiang Tian PhD and colleagues at the Institute for Systems Biology, Seattle, WA) and have excellent leads into heme?s role in regulating red cell differentiation and into the consequences of excessive heme from our preliminary investigations. We will also study ferroptosis, a recently described, poorly understood, non-apoptotic cell death pathway, involving P53 activation, decreased transcription of the cysteine/glutamate amino acid transporter SLC7A11, and an increased sensitivity to ROS. Since increased P53 activation also characterizes DBA, this mechanism would link our observations to other published data. We also have shown that slowing heme synthesis (or increasing heme export) improves the red cell production of Flvcr-deleted mice in vivo and DBA and del(5q) MDS patient marrow in vitro and will use this information to develop new therapeutic strategies for treating these disorders and potentially other anemias characterized by ineffective erythropoiesis.